Cell differentiation is a fundamental biological process. Central to it are the establishment of distinct programs of gene expression in the different cell types and the coordination of gene expression with morphological change. Formation of spores by Bacillus subtilis is a primitive system of cell differentiation that has become a paradigm for the study of cell differentiation in prokaryotes because of the ease of its genetic manipulation. All the key regulators of spore formation are also identified in all sequenced species of Bacillaceae, including the pathogens Bacillus anthracis and Clostridium difficile. Sporulation involves a characteristic division into two distinct cell types, the mother cell and the prespore. The prespore is engulfed by the mother cell and develops into the mature, resistant spore. Sporulation requires the action of four RNA polymerase sigma factors, sigmaF and then sigmaG in the prespore and sigmaE and then in sigmak the mother cell. The major objectives here are to understand how compartmentalized gene expression is established and maintained and how gene expression is coordinated with morphological change. Most of the proposal centers on B. subtilis. A series of interconnected lines of research will be pursued. It is proposed to investigate why sigmaG activity switches from the prespore to the mother cell in spollAdelta mutants. It is proposed to identify and characterize division genes and regulators of sF activation that are required for prespore-specific expression using a two-part compartmentalization test we have developed. It is proposed to investigate why certain sF-directed genes are poorly expressed when they are relocated near the chromosome terminus. It is proposed to identify and characterize genes involved in temporal control of sigmaF and sigmaG activity. It is proposed to investigate the establishment of compartmentalization in Sporosarcina ureae where the sporulation division is medially located, in contrast to its grossly asymmetric location for species of Bacillus and Clostridium.